Method for prophylaxis of infections in crops and ornamentals, preferably in viticulture, and in woody plants

ABSTRACT

The invention concerns a method for prophylaxis of infections by fungi, particularly by oomycetes, and of bacterial infections in crop and ornamental plants. Areas of application are vine, fruit, vegetable and ornamental plant growing. The method of the invention is characterized in that an aqueous solution of a protease, alone or in combination with ß-glucanases and/or chitinases, is prepared, stabilizers, stickers and wetters are added, and the additized solution is applied by conventional techniques to the plants a number of times within the vegetation period, preferably ahead of weather-related phases of high infection threat.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation of U.S. patent application Ser. No. 15/323,419,filed Jan. 1, 2017, which is the US National Stage of InternationalPatent Application No. PCT/DE2015/000289, filed Jun. 16, 2015, which inturn claims the benefit of German Patent Application No. 10 2014 009813.3, filed Jul. 3, 2014. The contents of the foregoing patentapplications are incorporated by reference herein in their entirety.

FIELD

The invention relates to a method for prophylaxis of infections causedby fungi, in particular oomycetes, as well as to prophylaxis ofbacterial infections in crops and ornamental plants. Areas ofapplication are vegetable, fruit, vine and ornamental plant growing,preferably vine and vegetable growing.

BACKGROUND

Relevance, Progress and Control of Infections Caused by Oomycetes

Plant diseases in crop and ornamental plants as well as in woody plantslead annually to high economic losses. Also, in hydroponic vegetable andornamental plant growing in greenhouses, fungi and oomycetes such asPhytophthora, Pythium and Peronospora play an important role as causefor plant diseases (Malathrakis & Goumas, 1999; Paulitz & Berlanger,2001). In vegetable (in particular potato and tomato), fruit, ornamentalplant and vine growing as well as in forestry, they are of particulareconomical relevance. In 2013, potatoes were globally grown at anagriculturally used area of 19.3 million hectare (Food and AgricultureOrganization of the United Nations, Statistics Division). The mostimportant pathogen in potato growing, which has gained even moreimportance by expanding plant growing into warmer climates, is theoomycete Phytophthora infestans, the pathogen that causes late blight(Oerke and Steiner, 1996). Its spreading is only controllable by theconstant use of fungicides (more than 235 million US $ per year only forpotato growing). The total market just for fungicides amounts to 5.5billion US $ per year (Powell & Jutsum, 1993).

In Germany, approximately 100 million euros are spent annually only forthe pest management in viticulture (Ochßner, 2009). In ecologicalviticulture, solely copper-containing plant protection products areused. However, these products are environmentally hazardous andpotentially toxic. For this reason, it is of great interest to establishalternative, improved means of protection that are effective againstpathogens and simultaneously ecologically friendly.

Here, the propagation cycle of oomycetes will be described using theexample of Plasmopara viticola, which causes grapevine downy mildew. Thelife cycle is divided into two sections of varying epidemiologicalsignificance. The oospore, which is important for the survival of thepathogen during winter, is formed in the sexual phase. During theasexual summer cycle, large quantities of sporangia are released.Plasmopara viticola hibernates as oospore in the soil in leaf debris ofheavily infested leafs. During late winter, the oospores becomegerminable and maintain their germination capacity until early summer.As soon as the soil warms up and sufficient precipitation has fallen,they germinate and form primary sporangia. Until mid-June, oospores keepgerminating during heavy rains. Some oospores may also be dormant formore than a year and germinate in the following year. Usually,germination and release of zoospores originates from the primarysporangium when temperatures rise above 10° C. and more than 8 mmprecipitation has fallen. Under these conditions, usually the firstyoung leaves of grapevine are unfolded, so that the primary infectioncan take place. For the primary infection by the germinated zoospores,the leaves have to be sufficiently wetted with water. Solely in thisphase, the infection can be prevented or reduced if damaging orinhibiting the zoospores has been successful.

The primary infection is the starting point of the summer cycle ofPlasmopara viticola, in which the pathogen reproduces asexually bysporangia and may cause epidemics if the conditions for reproducing arefavourable. The primary infection is followed by the incubation time, inwhich the pathogen matures inside the leaf without visible symptoms. Atreatment of the infection is no longer possible at that time. Growthand development of the pathogen are heavily dependent on temperature, sothat at higher temperatures the leaf tissue is faster penetrated by themycelium and the oil spots appear earlier in comparison to lowtemperatures. At the end of the incubation time, so-called oil spotsappear as a visible symptom of the fungal infection. As soon as at nightthe relative humidity rises above 95% and the temperatures remain above12° C., sporangiophores protrude from the stomata of the infectedlamina. The sporangia are spread by drops of water or movement of air.As soon as these drops of water contact a green part of their hostplant, the zoospores hatch. Hatching of zoospores and subsequentinfection occurs under optimal conditions at 24° C. within four hours.If the temperatures are lower or higher, hatching of zoospores isdelayed and the process of infection is prolonged. Plasmopara viticolamay infect leaves, inflorescence including stems, grapes and shoot tipsif they have stomata and if they are wetted. Small drops of water arealready sufficient for the infection, however, the conditions ofinfection are more favourable if the wetting with water is extensive andpersists for a long time. After each infection, again an incubation timefollows and, subsequently, sporangia will spread as soon as there issufficient humidity at night. Plasmopara viticola belongs to thepolycyclic pathogens and can undergo several propagation cycles duringone growing season. If optimal conditions for the spreading of sporangiaand for infections remain during longer periods and the incubation timesare short due to the temperature conditions, an epidemic can developrapidly. Drought delays the spread of Plasmopara viticola and impedesthe progression of epidemics. It is possible to predict locally phasesof high risk of an infection and, consequently, to take specificprophylactic preventive measures.

Under the climatic conditions prevalent in central Europe, infections bysuch pathogens are to be expected in every year. To what extent theseinfections lead to epidemics is highly dependent on the annual weatherconditions, and is not predictable at the beginning of the growingperiod. Epidemics, for example of grapevine downy mildew (Plasmoparaviticola), can become very severe in highly susceptible classical grapevarieties within a few rainy days. Therefore, this infection has to bedetected and controlled at an early stage. If the infestation is alreadyin an advanced stage, a later control is no longer possible. For thisreason, commercial plant growing is only possible with preventivemeasures against such infections. A forecasting method, which allowsperforming specifically preventive controls, was already developed forPlasmopara viticola at the German federal institute for viticulture(Staatliches Weinbauinstitut) and put into practice.

Currently, numerous fungicides are on offer for conventional plantgrowing. Solely in viticulture, 29 fungicides are approved forapplication in grapevine downy mildew at present.

For ecological vine growing, grapevine downy mildew is a challenge,since here a preventive treatment is indispensable and currently onlycopper-containing preparations (e.g. Cuprozin) are approved. Because ofthe known ecotoxicological concerns regarding copper, there is an urgentneed to find alternatives to this agent. These alternatives, however,have to have sufficient efficacy also under high infestation rates. Foryears, tests have demonstrated that the vast majority of preparationsthat are approved as plant strengthening agents do not show satisfactoryefficacy against grapevine downy mildew. Some plant strengthening agentsare effective against grapevine downy mildew at low infestation rates,however, a control measure would not have been necessary here. At ahigher infestation rates, which also justify combating from ancommercial point of view, the efficacy of the tested preparation wasinsufficient. From these tests it is perceived that no biologicalcontrol of grapevine downy mildew is practicable in ecological plantgrowing. Especially in ecological vine growing with the limitedpossibility to stop an epidemic, effective and practicable approachesfor the biological control of epidemics are urgently needed.

Relevance, Progress and Control of Bacterial Infections

Although the number of plant-pathogenic bacteria is lower than thenumber of fungi-like pathogens, the damage to crop plants caused bybacterial diseases is very high. Bacteria of the genus Xanthomonasglobally cause diseases in all main groups of higher plants, which areaccompanied by chlorotic and necrotic lesions, wilt and rots. An examplewith high economic relevance is black rot in varieties of cabbage, whichis caused by Xanthomonas campestris pv. campestris. Xanthomonas oryzaepv. oryzae leads to white leaves/bacterial blight of rice by infestationof rice plants, which is one of the most serious diseases in riceplants, and subsequently to major economic and social problems.Likewise, mention must be made of Pathovar X. axonopodis pv. citri, thepathogen that causes citrus cancer, and X. campestris pv. Vesicatoria,the pathogen that causes bacterial leaf spot disease on peppers andtomatoes which is of economic importance particularly in regions with awarm and humid climate. Furthermore, fire blight, caused by the pathogenErwinia amylovora, which is subject to mandatory reporting, has to bementioned. Host plants of E. amylovora are rosaceae such as apple, pearand quince. E. amylovora causes wilt of leaves and blossoms of infestedplant, which will then turn brown or black. Moreover, the bacterialspecies Pseudomonas syringae, which causes various plant diseases suchas cancer, wilt and spots in important crop plants such as tomato,pepper and soy bean, has to be mentioned. This widespread species is ofmajor importance in many plants grown under glass such as tomato,cucumber and courgette.

Most of the described bacterial plant pathogens belong to the group ofproteobacteria and are gram-negative organisms (e.g. Pseudomonas,Xanthomonas). However, there are also economically relevantgram-positive pathogens such as Clavibacter michiganensis ssp.Michiganensis, which causes bacterial wilt in tomatoes. This quarantinepest is of major importance in warmer and drier growing regions oftomatoes and in greenhouses.

Plant pathogenic bacteria have several strategies to survive in theenvironment, for example in soil, in plant material such as seeds or ininsects. Insects, other animals and humans play an important role intheir spreading. Water, e.g. in form of rain drops, is an importantvehicle in respect of the distribution at a plant. If bacteria aretransferred to a host plant, they penetrate through natural openings,such as stomata or hydathodes, through lesions of the plant. A highbacterial density as well as external conditions such as rain, highhumidity or damaged spots facilitate the infection of a plant. Bacteriacan easily multiply in the interior of the plant, they colonize theapoplast and damage from there the whole plant. They disturb thephysiology and morphology of plants and thus, they cause diseasesymptoms such as necrotic spots, defoliation, scabbing, wilt or rot (Dela Fuente and Burdman, 2011).

It is therefore crucial to protect crop plants against such bacterialinfections and thus to secure their harvest. Various chemical compoundsand copper compounds appear on the current list of plant protectionagents with an antibacterial effect that are approved in Germany. Coppercontaining preparations are the only means which, in turn, are allowedfor the use in ecological agriculture. Treatments with copper containingpreparations for the control of bacteriosis have a partial effect andshow their limitations as soon as the density of the bacterial inoculumpasses a certain threshold. Due to the known ecotoxicological effects ofcopper compounds and other agrochemicals, legitimate concerns about theuse of such plant protection products exist. Furthermore, even inGermany it is allowed in exceptional cases to use plant protectionagents that contain antibiotics such as streptomycin to control fireblight. In other countries, streptomycin is a legitimate means againstbacteriosis, but at the same time, the use of antibiotics is extremelyquestionable as undesirable effects on the environment as well asreductions in efficacy by development of resistance by bacteria may beencountered through undifferentiated use of antibiotics. Therefore, itis urgently needed to develop improved, highly effective andcommercially relevant alternatives to these agents that are moreenvironmentally-friendly and safer for the user.

Current Development of Strategies in Plant Protection

1. The demands on chemical plant protection products in respect ofefficacy, selectivity, specificity, biological degradation and efficacyon non-targeted organisms are steadily increasing. In the meantime, anumber of novel plant protection products is available that meet theserequirements. The use of numerous older compounds, such as hydrocarbons(aldrin, DDT, DDD, dieldrin, kelthane) is forbidden by now. Currentlyused chemical plant protection products, however, (e.g.ortho-phenylphenol E 231 or thiabendazole E 233) are becoming more andmore criticized. They show numerous harmful side effects, which maketheir use problematic. These include, the damage of the crop plantapart, changes in the taste of crops, toxic effects on numerousbeneficial organisms, development of resistant pest populations,incomplete decomposition by microorganisms and thus a too longpersistence and accumulation in the soil as well as finally leachinginto the groundwater and the accumulation in the food chain of humansand animals (Source: Umweltlexikon—www.umweltlexikon-online.de).

2. Increasingly, methods of biological and integrated plant protectionsuch as the use of beneficial organisms and pheromones against insects,the use of soil-borne bacterial und fugal antagonists as well as the useof plant extracts are being established. Among the most importantantagonistically acting classes of organisms are the bacteria Bacillus,Pseudomonas and Streptomyces and the fungi Trichoderma, Coniothyrium undVerticillium. Of particular importance in this context is the bacteriumBacillus subtilis that, as “plant growths promoting rhizobacterium”(PGPR), secretes phytosanitary metabolites, and the fungal genusTrichoderma, of which strains are used as “biocontrol agent” (Kücük, C.and M. Kivanc, 2002; DeMarco, J. L., et al., 2003). While many animalpests can be sufficiently controlled by these biological methods,infections caused by oomycetes are only difficult to combat. In theagricultural sector, the following pant diseases are of outstandingimportance due to their risk of infection and the resulting lossesthereof (Table 1):

TABLE 1 Tax. group Disease Agricultural area Examples Fungi AscomycotaPowdery Vegetable, grain, fruit, Erysiphe necator mildew wine andornamental (grapevine), Blumeria plant growing graminis (grain)Ascomycota Grey mold Vegetable, fruit, wine Botrytis cinerea andornamental plant (strawberry, grapevine, growing etc.) BasidiomycotaRusts Vegetable, grain, fruit Puccinia graminis and ornamental plant(grain), Phakospora growing pachyrhizi (soy bean) Basidiomycota SmutsVegetable, grain, fruit Ustilago maydis and ornamental plant (maize),Ustilago hordei growing (barley) Oomycetes Oomycetes Downy Vegetable,fruit, wine Phytophthora infestans mildew, late and ornamental plant(potato & tomato), blight growing Plasmopara viticola (grapevine)Bacteria Proteobacteria Fire blight, Vegetable, grain, fruit Erwiniaamylovorans, wilt, spots, and ornamental plant Pseudomonas syringae, andothers growing Xanthomonas campestris Actinomycetes Wilt and Vegetable,grain, fruit Clavibacter others and ornamental plant michiganensisgrowing

State of the Art

It is known that glycoside-cleaving enzyme preparations of the type ofnon-starch polysaccharide hydrolases are effective in the prophylaxisand therapy of plant pathogenic fungi. Here, a direct attack of theenzyme on the structures of the cell walls of the fungi, in particularof the oomycetes, is assumed (DE 10 2205 048 520, Biopract GmbH).However, these hydrolyses can also damage the cell wall of the plant andtherefore, they are only partially suitable for plant protection.

The use of enzymes of the type of non-starch polysaccharide hydrolasesfor prophylaxis and therapy of fungal phytopathogens is also supportedby a number of findings in other areas. For example, experience has beenacquired in combating oocytes-based fish mycosis with complex enzymepreparations from Trichoderma spp. (WO 2004/002574 A1 Biopract GmbH).

U.S. Pat. No. 6,663,860 (Tvedten, Dec. 16, 2003) describes proteases aspesticide against, inter alia, insects, bacteria and fungi. However, ause in prophylaxis of fungal infestation in viticulture is not intended.

Furthermore, the combination of a pesticide and an enzyme, for example aprotease, is described in various patent documents. Hereby, thedescribed effect is rather based on the pesticide and not on the addedenzyme alone (WO 2013/096383 A2, CN 103461383 A, WO 1997/047202 A1, WO1990/003732 A1). Other patent documents describe the combination ofdetergents and enzymes (U.S. Pat. No. 7,393,528 B2), of plant extractsand proteins (WO 2001/030161 A1) as well as of surface-active substancesand an enzyme (EP 184288 A1). Also these publications do not demonstratethat the enzyme itself is responsible for the pesticidal effect.

Finally, enzymes or enzyme combinations which show, inter alia, ananti-fungal or anti-bacterial effect, have been described in the past,for example a protease from plants (WO 1991/002459 A1), a protease froman earthworm (JP 2011177105A) or the culture supernatant of a Bacillusfermentation (JP 54073182 A).

None of the inventions mentioned above describes a comparably effectivesolution of the still existing problem of the infestation of crop plantsby oomycetes and bacteria. The proteases described here provide a highlyeffective and simultaneously environmentally sound alternative to commonplant protection products.

SUMMARY

The invention aims to develop a highly efficient means for controllinginfections caused by fungi, in particular oomycetes, and bacterialinfections in crop and ornamental plants that is harmless for the plantitself as well as for the ecosystem. The primary objective of thepresent invention is to develop a method for prophylaxis of infectionsof crop plants used in agriculture that are caused by phytopathogens. Inparticular, the problem of recognizing and preventing epidemics such asgrapevine downy mildew in wine varieties early shall be solved.Providing appropriate means is also comprised by the invention.

This problem is solved by the measures described in the claims. Themethod according to the invention is characterized in that a concentrateand a ready for use solution, respectively, are produced, which containa protease alone or a combination of proteases and β-glucanases and/orchitinases. The quintessence of the invention is the surprisingpossibility to provide proteases alone as effective means forcontrolling infections in crops and ornamental plants.

Furthermore, the protective products can contain stabilizers, adhesiveand wetting agents, which improve the application properties. Commonrain stabilizers and UV stabilizers can also be included in thesemixtures.

This mixture is applied by conventional application systems at fixedtimes, which are determined based on the weather condition, in a waythat the whole plant is wetted. The application can be effected attemperatures between 4° C. and 34° C. and therefore during the wholegrowing season. In plant cultivation under glass, the application islargely independent from the weather conditions and temperatures rangefrom 15° C. to 25° C. This mode of application ensures that the enzymepreparations are active and an infection of the plant by e.g. zoosporesof phytopathogenic oomycetes or bacterial pathogens such as Pseudomonassyringae is prevented. The applied quantity per area has to bedetermined dependent on the crop plant. For example in vine growing,approximately 400 to 800 liter spray cocktail are currently applied onan area of one hectare. The described enzyme preparations are mixed in away that conventional spraying technique can continued to be used.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Leaf discs treated with water (A), copper-containing plantprotection product (B), protease I (C), protease II (D) or protease III(E).

FIG. 2: Efficacy test of Prot III at potted Müller-Thurgau vines.Presented is the degree of infestation of P. viticola in the treatedvariants protease III 0.01% to 0.5% in comparison to the internalstandards water control and copper reference solution. The degree ofinfestation was effectively reduced by using Prot III. The increasedinfestation of plants treated with 0.5% Prot III (*) was caused by aspray shadow on a single leaf. The degree of infestation was calculatedbased on the proportional infestation of 6 individual plants with up to6 leaves per variant.

FIG. 3: Presentation of the inhibiting effect of protease III on thegrowth of C. michigenesis. Presented are dilution series of a bacterialculture of different concentrations of protease III. Numbers below theagar plates indicate the dilution up to which the bacteria grew.

FIG. 4: Proliferation of P. syringea bacteria in foliar segments oftomato within 21 days after inoculation. Tomato plants were mock-treated(blank) and sprayed with a protease III or protease III in Nufilm-P,respectively. The colony forming units (CFU) were isolated from 0.7 cm²foliar segments and counted after an incubation of 48 hours.

DETAILED DESCRIPTION

The invention described constitutes a significant progress in comparisonto currently established means and methods.

The advantages over the state of the art are described in the following:

-   -   In contrast to copper-containing preparations or other chemical        plant protection products, the use of enzyme preparations is        harmless for the ecosystem since the agent is completely        degraded in the soil and not accumulated. Therefore,        considerable environmental damages are prevented.    -   There are no phytotoxic reactions, since the applied proteases        according to the invention do not attack the plant surface.    -   Proteases and other enzymes are effective during plant growth.        They do not adhere at one location to the leaf structure but        disperse in a liquid film on the surface.    -   The efficacy of the enzyme remains intact for a relevant period        of several days despite of rain and UV radiation. This stability        can be improved by suitable formulations if necessary.

Proteases, also called peptidases, cleave peptide bonds in proteins andso promote their degradation in peptides or amino acids. Proteases aredivided into the following groups based on their mode of action: serineproteases, EC 3.4.21.-, (S), cysteine proteases (C), aspartic protease(A), metalloproteases (M), and unknown or so far unclassified proteases(Handbook of Proteolyse Enzymes, A. J. Barrett, N. D. Rawlings, J. F.Woessner (eds), Academic Press (1998)).

Proteases that are used according to the invention described are inparticular serine proteases. The catalytic mechanism of this enzymeclass is based on the nucleophilic hydroxyl group of the amino acidserine that can cleave peptide bonds. Respective enzymes can be gainedfrom culture supernatants for example from microorganisms of the generaNocardiopsis or Bacillus. The respective enzymes can also be producedrecombinantly. Moreover, the effective proteases can be mutants,variants or fragments of the described enzymes that act analogous.

The activity of the proteases can be determined with every detectionmethod in which a substrate is used that contains the respective peptidebonds (e.g. casein).

Surprisingly, it has been found that protease preparations, which areused for instance in animal feeding, prevent the infection of plants byphytopathogenic oomycetes and bacteria. Particularly zoospores thatarise during the propagation cycle of oomycetes and that are responsiblefor the actual infection of the leaf tissue, are damaged irreversibly bythe activity of these enzymes, and the infection of the so protectedplant does not occur. The mode of action against bacterial infestationhas not been elucidated so far. The significant impact of these enzymeswas not expected to such extent since the modes of action and points ofattack do not correspond to the mode of action described forβ-glucanases or chitinases. The protective effect can be increased by acombination with β-glucanases and/or chitinases.

Commercially available preparations, which contain the proteasesdescribed, are for example Ronozyme®ProAct® (DSM Nutritional ProductsAG, examples 1-9: Prot III), which contains a serine protease fromNocardiopsis sp., or Alcalase® (Novozymes AG), which contains primarilyone serine protease, Subtilisin A, from Bacillus licheniformis.Furthermore, select protease preparations that show a protective effectare those by the company Lumis Enzymes (PAP 2XS), which, as far as isknown, contains papain from papaya, by the company Cyadic (ProteasePlus, Protease AP Conc) and by the company AB Enzymes (BIOTOUCH ROC250LC), which contains, as far as is known, a protease from Trichoderma.

Glucanases and chitinases are enzymes that can hydrolyze glucans orchitin. They are assigned to the enzyme class E.C. 3.2.1.-, whichcomprises glycosidases, i.e. enzymes that cleave O- and S-glycosidicbonds.

Following the characteristics of the specific disease, the enzymeagainst the leaf pathogens (e.g. downy mildew or Pseudomonas syringae)are applied by treating the aerial parts of the plant (e.g. by spraying)with concentrations of an enzyme preparation of 0.001% to 1%. Proteasesand glycosylases are preferably used in different mixing ratios of thesingle enzymes.

The effect, according to the invention, of the enzyme preparations,which manifests itself in preventing the occurrence of the infection, isachieved by using proteases alone or as a mixture.

The enzymes are gained from culture supernatants of microorganisms. Thecomponents for the inactivation of the pathogen are preferably used inan aqueous milieu with a pH ranging from 4.5 to 8.5, preferably a pHranging from 6.0 to 7.5. They are used at a water temperature of 4° C.to 34° C., preferably at 10° C. to 25° C.

In the following, the invention shall be illustrated by examples. Theexamples 1-7 demonstrate the use of proteases as protection againstoomycetes while the examples 8 and 9 describe the protective effectagainst bacterial infections. Ronozyme®ProAct® (DSM Nutritionai ProductsAG) was used as protease III.

EXAMPLES

Protecting Crop Plants from Infection Caused by Omycetes

Example 1

Infection Suppressing Effect of Punctual Foliar Application of ProteinCleaving Enzyme Preparations Against Plasmopara viticola in Leaf Discs.

Leaf discs of the grapevine Vitis vinifera cv. Müller-Thurgau weretreated once with different protein cleaving enzyme preparations(protease I, II and III) by spray application, so that the bottom sideof the leaf of the used leaf discs was evenly wetted. The preparationscontained a serine protease each, which was gained either from a speciesof the genus Nocardiopsis or Bacillus. The enzyme preparations used forthe treatment were tested in concentrations ranging from 0.01% to 1%(v/v). The pH value of the preparations diluted in water ranged from 6to 7.5. As control, the leaf discs were either sprayed with acopper-containing plant protection product or with water. 24 hours aftertreatment, the artificial infection of the leaf discs was performed withPlasmopara viticola (approximately 40000 spores per ml water), thepathogen that causes downy mildew in grapevine. Subsequently, the leafdiscs were incubated on water agar plates at 22° C. for six days in aplant chamber with day-night rhythm.

The degree of infestation is calculated from the ratio of the total areaof the leaf and the infected area of the leaf. For the analysis, imageevaluation software was used, which distinguished between the total area(number of green pixel of the leaf discs at the beginning of theexperiment) and the infected area (number of white pixels at the end ofthe experiment). Two of the tested protease preparations (I, III) aswell as the copper-containing plant protection product (Cuprozin)prevented the infestation and the development of Plasmopara viticolaeffectively (0% infestation), the third preparation, protease II,prevented infestation only to some extent (38% infestation). However,leaf discs that were sprayed with water showed markedly infestation (seeTable 2 and FIG. 1).

TABLE 2 Degree of infestation (%) calculated from the ratio of the totalarea of the leaf disc in relation to the infected area per leaf discafter treating the leaf discs with three different protease preparations(n = 36) Degree of infestation (MW) Standard deviation H₂O 87% 6%Cuprozin 0% 1% Protease I 0% 1% Protease II 38% 28% Protease III 0% 0%

Example 2

Infection Suppressing Effect of Punctual Foliar Application ofCombinations of Proteases, Chitinases and Glycoside-Cleaving EnzymePreparations Against Plasmopara viticola in Leaf Discs.

Leaf discs of the grapevine Vitis vinifera cv. Müller-Thurgau weretreated once with an enzyme combination consisting of protease,chitinase and β-glucanase in a mixing ration of 1:1:1 by sprayapplication, so that the bottom side of the leaf of the used leaf discswas evenly wetted. The concentration of the enzyme preparations used was0.1% (v/v) for each enzyme. The pH value of the in water dilutedpreparations ranged from 6 to 7.5. As control, the leaf discs wereeither sprayed with a copper-containing plant protection product or withwater. 24 hours after treatment, the artificial infection of the leafdiscs was performed with Plasmopara viticola (approximately 40000 sporesper ml water), the pathogen that causes downy mildew in grapevine.Subsequently, the leaf discs were incubated on water agar plates at 22°C. for six days in a plant chamber with day-night rhythm.

The degree of infestation is calculated from the ratio of the total areaof the leaf and the infected area of the leaf. For the analysis, imageevaluation software was used which distinguished between the total area(number of green pixels of the leaf discs at the beginning of theexperiment) and the infected area (number of white pixels at the end ofthe experiment). The development of Plasmopara viticola was effectivelyprevented on the leaf discs that were treated with the enzymes and thecopper-containing plant protection product.

Example 3

Infection Suppressing Effect of Punctual Foliar Application of ProteinCleaving Enzyme Preparations Against Plasmopara viticola in HothousePlants.

Young vines of the variety Vitis vinifera cv. Müller-Thurgau wereentirely single treated with a protein cleaving enzyme preparation(protease III) by means of a stationary application unit. Theconcentrations of the enzyme preparations used were 0.1%, 0.2% and 0.5%(v/v). The pH values of the spraying cocktails were adjusted between 6.5and 7.5. As control, the further potted vines were either sprayed with acopper-containing plant protection product or with water. 24 hours aftertreatment with the protease preparation, the artificial infection of theleaves was performed with Plasmopara viticola, the pathogen that causesdowny mildew in grapevine. Subsequently, the plants were incubated forone week in a greenhouse at 20° C. The infestation was determined byvisual observation and given as the proportion (%) of diseased andnecrotic alterations, respectively, at leaves and stems in relation tothe total mass of the plant per replication (100%) and documentedphotographically. A scoring scale with the graduations 1, 5, 10, 15, 20,25, 30, 40, 50, 90, 100% diseased alterations was used.

The development and spread of Plasmopara viticola was effectivelyprevented on the plants that were treated with protease III andcopper-containing plant protection products, while leaves that weresprayed with water showed high infestation (FIG. 2).

Example 4

Infection Suppressing Effect of Periodic Application of Protein CleavingEnzyme Preparations Against Plasmopara viticola in Field Trials.

Whole vines of the variety Vitis vinifera cv. Blauer Spätburgunder weresprayed by means of a tunnel spraying machine with an enzyme-cleavingenzyme preparation (Prot III) during the growing season repeatedly atintervals of 8 to 14 days, so that the surface of the vines was evenlywetted. The concentration of the enzyme preparation used was 0.1% (v/v).The pH values of the spraying cocktails were adjusted between 6.5 and7.5. For improved wetting of the leaves, a wetting agent (TREND 90) wasfurther added.

At the end of the growing season, the degree of infestation andinfestation frequency by downy mildew at leaves and grapes were scored.The development of Plasmopara viticola was effectively prevented atvines in open land.

Example 5

Infection Suppressing Effect of Protein Cleaving Enzyme PreparationsAgainst the Pathogen that Causes Late Blight (Phytophtora infestans) inTomato Plants

Tomatoes of the variety Red robin were sprayed at 4-leaf stage with theprotease preparation (protease III, 0.1% (v/v)). The pH values of thespraying cocktails were adjusted between 6.5 and 7.5. As additionalvariants, common wetting agents (T/S forte, Biomaxima, Nufilm) wereadded with a concentration of 0.02% (v/v) to the protease solution. Thecommercial copper preparation Atempo and water served as internalcontrols. Per variation, 5 replicates with one plant each were prepared.

24 hours after applying the enzymes, the artificial inoculation with thepathogen Phytophtora infestans was performed with a sporangiaconcentration of 80 000 spores per ml. For each plant, 6 ml suspensionwere applied. The plants were placed in the incubator at approximately16° C. and 100% relative humidity without light. After 24 hours, alighting rhythm of 16:8 hours was set and the humidifier was turned off.Scoring was performed 6 days after infection. The infestation wasdetermined by visual observation and given as proportion (%) of diseasedor necrotic alterations at leaves and stems in relation to the totalmass of the plant and documented photographically (Tab. 3).

TABLE 3 Infestation by Phytophtora infestans and efficiency of theprotease preparation Infestation [%] Mean No. Variant (Standarddeviation) Efficiency [%] 1 Protease III 12.00 (2.74) 87.50 2 ProteaseIII + T/S-Forte 18.00 (2.74) 81.25 3 Protease III + BioMaxima 11.00(5.48) 88.54 4 Protease III + Nufilm P 12.00 (2.74) 87.50 5 Atempo(copper-containing  1.80 (1.79) 98.13 reference) 6 Water control 96.00(5.48) 0.00

Example 6

Infection Suppressing Effect of Protein Cleaving Enzyme PreparationsAgainst Pseudooeronospora cubensis in Cucumber Plants

Cucumber plants were grown in a climatic chamber. For proving theprotective effect of proteases, the bottom sides of the leaves weresprayed with approximately 6 ml of the protease preparation (Prot III),which was an aqueous solution with a concentration of 0.1%. The pHvalues of the spraying cocktails were adjusted between 6.5 and 7.5. Acommon copper preparation (Cuprozin Progress) and water served ascontrols. Per variation, 6 replicates with one plant each were prepared.One day after applying the enzymes, the trial plants were infected withPseudoperonospora cubensis (75 000 spores per ml). Incubation wasperformed at room temperature in a greenhouse at a relative humidity ofmore than 95%. During the first 48 hours, the plants were incubated inthe dark, then, the plants were kept in a day-night rhythm of 16/8hours. Scoring was performed 10 days after infection. Thereby, theproportional infestation of the plants was determined. Infestation couldbe reduced to less than 4% by using the preparation Prot III (Tab. 4).

TABLE 4 Infestation [%] Mean (Standard Efficiency No. Variant deviation)[%] 1 Protease III 3.6 (0.4) 94.1 5 Cuprozi Progress (copper-containing9.0 (4.7) 85.2 standard) 6 Water 60.4 (11.9) 0.00

Example 7

Comparison of the infection Suppressing Effect of Different ProteasePreparations Against Pseudooeronospora cubensis in Cucumber Plants

Cucumber plants were grown in a climatic chamber. For the comparison ofthe efficiency of different protease preparations, the bottom sides ofthe leaves were sprayed with approximately 6 ml of each proteasepreparation (Prot III-Prot IX), which was an aqueous solution with aconcentration of 0.1%. The pH values of the spraying cocktails wereadjusted between 6.5 and 7.5. A common copper preparation (CuprozinProgress) and water served as controls. Per variation, 6 replicates withone plant each were prepared. One day after applying the enzymes, thetrial plants were infected with Pseudoperonospora cubensis. Incubationwas performed at room temperature in a greenhouse at a relative humidityof more than 95%. During the first 48 hours, the plants were incubatedin the dark, then, the plants were kept in a day-night rhythm of 16/8hours. Scoring was performed 10 days after infection. Hereby, theproportional infestation of the plants was determined. The efficiency ofthe individual preparations is presented in table 5. The preparationwith the best effect was protease III. The proteases IV, VIII and IXhave a similar effect. As far as known, the organisms of which therespective protease originated, is stated.

TABLE 5 Overview of the protease preparations that were used in theexperiment as well as the infestation and the respective efficienciesInfestation [%] Mean (Standard Efficiency Sample ID Origin Productdeviation) [%] Prot III Nocardiopsis Ronozyme 0.4 (0.4) 98.6 ProAct ProtIV Bacillus Alcalase 0.5 (0.4) 98.3 Prot V Bacillus Savinase 9.8 (4.8)65.8 Prot VI Aspergillus Flavourzyme 35.0 (21.5) — Prot VIII Papaya PAP2XS 0.8 (0.3) 97.2 Prot IX Bacillus Protease AP 1.0 (0.6) 98.5 ConcCopper- — Cuprozin 7.4 (6.2) 74 containing fungicide Progress

Protecting Crop Plants from Infection Caused by Bacteria

Example 8

Plating Experiment for Proving the Growth Inhibiting Effect of ProteasesAgainst Clavibacter michiganensis

A culture of Clavibacter michiganensis was grown until late log phaseand accordingly diluted to OD_(600 nm)=1.0 in a 10 nM NaCl solution.This starting culture was plated in 12 dilutions ranging from 10⁻¹ to10⁻¹² on nutrient agar each which contained the preparation protease IIIin concentrations of 0.01-1%. Two control plates were free of proteaseIII and showed the maximal growth of Clavibacter michiganensis at thegiven conditions (FIG. 3, left: 10⁻⁵). The bacterial growth waseffectively inhibited at protease concentrations starting at 0.05% sinceonly the most concentrated dilutions were grown (10⁻¹ to 10⁻³, see FIG.3). The high potential of protease III as plant protection means forcontrolling bacterial wilt in tomato (Clavibacter michiganensis subsp.michiganensis) becomes apparent by this experiment.

Example 9

Protecting Tomato Plants from Infections Caused by Pseudomonas syringae

For this experiment, tomato plants of the variety “Red Robin” weresprayed with a 0.1% protease solution (protease III) with and withoutthe addition of an adhesive agent (NufilmP) and incubated at 22° C. for24 hours. The pH value of the spraying cocktail was adjusted between 6.5and 7.5. As controls served 4 plants each that were sprayed with tapwater and a mock solution, respectively. 24 hours after applying theenzymes, plants were infected with Pseudomonas syringae.

The first samples of 0.7 cm² large foliar segments were taken two hoursafter inoculation, further samples were taken at day 7, 14 and 21 afterinoculation. For analysis 4 different single leaves were taken from eachof 4 different plants, and from each of these four different singleleaves 4 foliar segments were taken for analysis. At the beginning, thenumber of colony forming units (CFU) per foliar segment was 1×10³. Inthe control (water), the number of CFU increased to 1×10⁶ CFU per foliarsegment within three weeks. Plants that were treated with protease IIIremained at a constant level (10³ CFU per leaf) in the first two weeks.After three weeks, the number of CFU per leaf on leaves treated withprotease was significantly decreased to 10 CFU per foliar segment (FIG.4). The protease used was considerably more effective than the mocksubstance.

LITERATURE

-   DE LA FUENTE, L. and BURDMAN, S. 2011. Pathogenic and beneficial    plant-associated bacteria. In Agricultural Sciences, [Ed. Rattan    Lal], in Encyclopedia of Life Support Systems (EOLSS), Developed    under the Auspices of the UNESCO, Eolss Publishers, Oxford, UK,    [http://www.eolss.net]-   DE MARCO, J. L; VALADARES-INGLIS, M. C. and C R. FELIX, 2003:    Production of hydrolytic enzymes by Trichoderma isolates with    antagonistic activity against Crinipellis perniciosa, the causal    agent of witches' broom of cocoa. Brazilian J. Microbiol. 34, 33-38-   KASSEMEYER H.-H. (2004) Forschungsvorhaben für das Programm des    Bundesministeriums für Verbraucherschutz, Ernährung und    Landwirtschaft zur Förderung von Forschungs—und Entwicklungsvorhaben    sowie zum Technologie—und Wissenstransfer im ökologischen Landbau.    “Innovationen zur Verbesserung der Rahmenbedingungen für den    ökologischen Weinbau. Erarbeitung von wissenschaftlichen Ansätzen    zur biologischen Kontrolle der Rebenperonospora und für Strategien    zu deren Regulierung im ökologischen Weinbau”, Projektnummer    020E269, Staatliches Weinbauinstitut Freiburg-   KUDO, S. and C. TESHIMA, 1991: Enzyme activities and antifungal    action of fertilization envelope extract from fish eggs. The Journal    of Experimental Zoology 259, 392-398-   KUDO, S., 1992: Enzymatic basis for protection of fish embryos by    the fertilization envelope. Experientia 48, 277-281-   KUDO, S., 2000: Enzymes responsible for the bactericidal effect in    extracts of vitelline and fertilization envelopes of rainbow trout    eggs. Zygote 8, 257-265-   KÜCÜK, C. and M. KIVANC 2002: Isolation of Trichoderma spp. and    determination of their antifungal, biochemical and physiological    features. Türk. J. Biol. 27, 247-253-   MALATHRAKIS, N. E., GOUMAS, D. E. 1999: Fungal and bacterial    diseases. See Ref. 4 pp. 34-47.-   MÜNCH, S., NEUHAUS, J. M., BOLLER, T., KEMMERLING, B. and K. H.    KOGEL 1997: Expression of ß-1,3-glucanase and chitinase in healthy,    stem rust-affected and elicitor-treated near-isogenic wheat lines    showing Sr5 or Sr24-specific rust resistance. Planta 201, 235-244-   OERKE, E. CH. und U. STEINER 1996: Ertragsverluste und    Pflanzenschutz: Die Anbausituation für die wirtschaftlich    wichtigsten Kulturen. Schriftenreihe der deutschen    Phytomedizinischen Gesellschaft, Eugen Ulmer GmbH & Co., Stuttgart-   PAULITZ, T. C. BELANGER, R. R. 2001: Biological control in    greenhouse Systems. Annu. Rev. Phytopathol. 39, 103-133-   POWELL, K. A., JUTSUM, A. R. (1993) Technical and commercial aspects    of biocontrol products. Pestic. Sei. 37, 315-321.-   SCALA F., S. L. WOO, I. GARCIA, A. ZOINA, E. FILIPPONE, J.-A.    PINTOR-TORO, G. DEL SORBO, B. ALOJ and M. LORITO. 1998. Transgenic    tobacco and potato plants expressing antifungal genes from    Trichoderma are resistant to several plant pathogenic fungi. 7th    International Congress of Plant Pathology, August 9-16 1998,    Edinburgh, Scotland, Offered Papers Abstracts—Volume 3: 5.3.10.-   WO 2004/002574 A1 Biopract GmbH, Berlin; LEIBNIZ-Institut für    Gewässerökologie und Binnenfischerei im Forschungsverbund Berlin    e.V. Verfahren zur Prophylaxe und Therapie von Mykosen bei Fischen    und Wirbellosen und deren Entwicklungsstadien. (expired)-   DE 10 2205 048 520 Biopract GmbH, Berlin GmbH, Institut für    Gemüse—und Zierpflanzenbau Großbeeren/Erfurt. Verfahren zur    Prophylaxe und Therapie von Mykosen bei Nutz—und Zierpflanzen sowie    bei Gehölzen, insbesondere in hydroponischen Systemen, Jul. 10, 2007

We claim:
 1. A method for prophylaxis of infection by Phytophthorainfestans or Pseudomonas syringae, in crops and ornamental plants,comprising spraying plants with an aqueous solution of a bacterialserine protease, characterized in that said bacterial serine protease isderived from Bacillus sp.
 2. The method according to claim 1,characterized in that said aqueous solution of a bacterial serineprotease for the control of pathogens is applied at a dosage of 0.001%to 1%.
 3. The method according to claim 1, characterized in that a timeinterval treatment of the plants is performed by spraying aerial partsof the plants.
 4. The method for according to claim 1, characterized inthat said aqueous solution of a bacterial serine protease is applied ata pH of 4.0 to 8.0.
 5. The method according to claim 1, characterized inthat said aqueous solution of a bacterial serine protease is applied attemperatures of 4° C. to 34° C.
 6. The method according to claim 1,characterized in that said aqueous solution of a bacterial serineprotease is formulated with adhesive and wetting agents as well asstabilizers.
 7. The method according to claim 1, characterized in thatsaid aqueous solution consists essentially of said bacterial serineprotease.
 8. The method according to claim 1, characterized in that saidaqueous solution consists of a combination of bacterial serineproteases.